DNA sequence and preparation of grass pollen allergen Phl p 4 by recombinant methods

ABSTRACT

The present invention relates to the preparation and use of recombinant variants of group 6 allergens of the  Poaceae  (true grasses), which are characterized by reduced IgE reactivity compared with known wild-type allergens and at the same time substantially retained reactivity with T-lymphocytes.

BACKGROUND OF THE INVENTION

The present invention relates to the provision of the genetic sequenceof the major grass pollen allergen Phl p 4. The invention also coversfragments, new combinations of partial sequences and point mutantshaving a hypoallergenic action. The recombinant DNA molecules and thederived polypeptides, fragments, new combinations of partial sequencesand variants can be utilized for the therapy of pollen-allergicdiseases. The proteins prepared by recombinant methods can be employedfor the in vitro and in vivo diagnosis of pollen allergies.

Type 1 allergies are of importance worldwide. Up to 20% of thepopulation in industrialized countries suffers from complaints such asallergic rhinitis, conjunctivitis or bronchial asthma. These allergiesare caused by allergens present in the air (aeroallergens) which areliberated from sources of various origin, such as plant pollen, mites,cats or dogs. Up to 40% of these type 1 allergy sufferers in turnexhibit specific IgE reactivity with grass pollen allergens (Freidhoffet al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001).

The substances which trigger type 1 allergy are proteins, glycoproteinsor polypeptides. After uptake via the mucous membranes, these allergensreact with the IgE molecules bonded to the surface of mast cells insensitized individuals. If two IgE molecules are cross linked to oneanother by an allergen, this results in the release of mediators (forexample histamine, prostaglandins) and cytokines by the effector celland thus in the corresponding clinical symptoms.

A distinction is made between major and minor allergens depending on therelative frequency with which the individual allergen molecules reactwith the IgE antibodies of allergy sufferers.

In the case of timothy grass (Phleum pratense), Phl p 1 (Petersen etal., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthiesenand Lowenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al.,1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen etal., 1995, Int. Arch. Allergy Immunol. 108, 49-54). Phl p 2/3 (Doleceket al., 1993, FEBS 335 (3), 299-304), Phl p 4 (Haavik et al., 1985, Int.Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int.Arch. Allergy Immunol. 97: 287-294, Fischer et al., 1996, J. AllergyClin. Immunol. 98: 189-198) and Phl p 13 (Suck et al., 2000, Clin. Exp.Allergy 30: 324-332; Suck et al., 2000, Clin. Exp. Allergy 30:1395-1402) have hitherto been identified as major allergens.

Phl p 4 has been mentioned as a basic glycoprotein having a molecularweight of between 50 and 60 kDa (Haavik et al., 1985, Int. Arch. AllergyAppl. Immunol. 78: 260-268). The Phl p 4 molecule is trypsin-resistant(Fischer et al., 1996, J. Allergy Clin. Immunol. 98: 189-198), and70-88% of grass pollen allergy sufferers have IgE antibodies againstthis molecule (Valenta et al., 1993, Int. Arch. Allergy Immunol. 97:287-294; Rossi et al., 2001, Allergy 56:1180-1185; Mari, 2003, Clin.Exp. Allergy 33:43-51). Homologous molecules have been described fromrelated grass species (Su et al., 1991, Clin. Exp. Allergy 21: 449-455;Jaggi et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 342-348; Jaggiet al., 1989, J. Allergy Clin. Immunol. 83: 845-852; Leduc-Brodard etal., 1996, J. Allergy Clin. Immunol. 98:1065-1072; 14-17). Thesehomologous molecules of the Poaceae form allergen group 4, whosemolecules have high immunological cross-reactivity with one another bothwith monoclonal mouse antibodies and with human IgE antibodies(Fahlbusch et al., 1993 Clin. Exp. Allergy 23:51-60; Leduc-Brodard etal., 1996, J. Allergy Clin. Immunol. 98:1065-1072; Su et al., 1996, J.Allergy Clin. Immunol. 97:210; Fahlbusch et al., 1998, Clin. Exp.Allergy 28:799-807; Gavrovi-Jankulovi et al., 2000, Invest. Allergol.Clin. Immunol. 10 (6): 361-367; Stumvoll et al. 2002, Biol. Chem. 383:1383-1396; Grote et al., 2002, Biol. Chem. 383: 1441-1445; Andersson andLidholm, 2003, Int. Arch. Allergy Immunol. 130: 87-107; Mari, 2003,Clin. Exp. Allergy, 33 (1): 43-51).

In contrast to the above-mentioned major allergens of Phleum pratense(Phl p 1, Phl p 2/3, Phl 5a and 5b, Phl p 6 and Phl p 13), the primarystructure of Phl p 4 has not yet been elucidated. Likewise, there is nocomplete sequence of molecules from group 4 from other grass species.

The determination of the N-terminal amino acid sequence was hithertounsuccessful. However, the causes of this are not known. Fischer et al.(J. Allergy Clin. Immunol., 1996; 98: 189-198) assume N-terminalblocking, but were able to purify an internal peptide after degradationwith lysyl endopeptidase and to determine its sequence: IVALPXGMLK (SEQID NO: 7).

This peptide has homologies to peptide sequences in the ragweedallergens Amb a1 and Amb a2 and similarities to sequences in proteinsfrom maize (Zm58.2), tomato (lat 59, lat 56) and tobacco (G10) (Fischeret al., 1996, J. Allergy Clin. Immunol. 98: 189-198). For Loliumperenne, peptide fragments having the following sequence have beendescribed for the basic group 4 allergen: FLEPVLGLIFPAGV (SEQ ID NO: 8)and GLIEFPAGV (SEQ ID NO: 9) (Jaggi et al., 1989, Int. Arch. AllergyAppl. Immunol. 89: 342-348).

Peptides have likewise been obtained from the group 4 allergen fromDactylus glomerata by enzymatic degradation and sequenced: DIYNYMEPYVSK(P15, SEQ ID NO: 10), VDPTDYFGNEQ (P17, SEQ ID NO: 11),ARTAWVDSGAQLGELSY (P20, SEQ ID NO: 12) and GVLFNIQYVNYWFAP (P22, SEQ IDNO: 13) (Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072).

Peptides have also been obtained from the group 4 allergen ofsubtropical Bermuda grass (Cynodon dactylon) by proteolysis andsequenced: KTVKPLYIITP (S, SEQ ID NO: 14), KQVERDFLTSLTKDIPQLYLKS (V49L,SEQ ID NO: 15), TVKPLYIITPITAAMI (T33S, SEQ ID NO: 16),LRKYGTAADNVIDAKWDAQGRLL (T35L, SEQ ID NO: 17), KWQTVAPALPDPNM (P2, SEQID NO: 18), VTWIESVPYIPMGDK (V26L, SEQ ID NO: 19), GTVRDLLXRTSNIKAFGKY(L25L, SEQ ID NO: 20), TSNIKAFGKYKSDYVLEPIPKKS (T22L, SEQ ID NO: 21),YRDLDLGVNQWG (P3, SEQ ID NO: 22), SATPPTHRSGVLFNI (V20L, SEQ ID NO: 23),and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (V14L, SEQ ID NO: 24) (Liaw etal., 2001, Biochem. Biophys. Research Communication 280: 738-743).

However, these described peptide sequences for Phl p 4 and group 4allergens have hitherto not resulted in the elucidation of the completeprimary structure of group 4 allergens.

The object on which the present invention is based therefore comprisedthe provision of the complete DNA sequence of Phl p 4 and of acorresponding recombinant DNA on the basis of which the Phl p 4 allergencan be expressed as protein and made available for pharmacologicallysignificant utilization as such or in modified form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Internal DNA sequence (SEQ ID NO: 25) of the Phl p 4 geneAmplicons obtained with genomic DNA were cloned with the degeneratedprimers No. 30 (sense) and No. 37 (antisense), both shown in italics,and sequenced. The sequence shown represents the consensus from 6clones. The specific sense primer No. 82 created from this sequence isshown underlined.

FIG. 2: 3′ end of the nucleic acid sequence (SEQ ID NO: 26) of the Phl p4 gene.

Amplicons were obtained with the specific sense primer No. 82 (shown initalics) and an anchor primer in a 3′-RACE PCR with Phleum pratense cDNAand sequenced. The sequence shown represents the consensus from 3sequencing processes and covers the 3′ end of the Phl p 4 gene to thestop codon (double underlined). The sequence ranges employed forconstruction of the antisense primers No. 85 and No. 86 are shownunderlined.

FIG. 3: Localization of the Phl p 4 peptides in the deduced amino acidsequence of the Phl p 4 allergen (SEQ ID NO: 2). The peptides P1-P6 (SEQID NOs: 27-32) obtained from the amino acid sequencing of the purifiedand fragmented Phl p 4 allergen can unambiguously be assigned to theamino acid sequence of the Phl p 4 gene derived from the nucleic acidsequence.

FIG. 4: Determination of the identity of recombinant Phl p 4 (rPhl p 4)by means of monoclonal antibodies 5H1 (blot A) and 3C4 (blot B) specificfor nPhl p 4 by Western blot. Track 1: E. coli total cell extractcomprising rPhl p 4 fragment 1-200 Track 2: E. coli total cell extractcomprising rPhl p 4 fragment 185-500 Track 3: E. coli total cell extractcomprising rPhl p 4 Track 4: purified nPhl p 4 from Phleum pratense (←):termination or degradation fragments of C-terminal rPhl p 4 fragment orrPhl p 4 entire molecule.

FIG. 5: Determination of the reactivity of recombinant Phl p 4 (rPhl p4) using IgE from sera of grass pollen allergy sufferers by Westernblot. Extracts of transformed E. coli cells which either express thecomplete Phi p 4 gene or the N-terminal fragment 1-200 or the C-terminalfragment 185-500 were separated in the SDS-PAGE and transferred tonitrocellulose membranes. The blot was incubated with sera from grasspollen-allergic donor A, B or C, and bound IgE was subsequently detectedcalorimetrically via an anti-human IgE antibody conjugated with alkalinephosphatase. Track 1: E. coli total cell extract comprising rPhl p 4fragment 1-200 Track 2: E. coli total cell extract comprising rPhl p 4fragment 185-500 Track 3: E. coli total cell extract comprising rPhl p 4Track 4: purified nPhl p 4 from Phleum pretense.

The numbers used above and below for nucleotide or amino acid sequences“SEQ ID NO” relate to the sequence protocol attached to the description.

DESCRIPTION OF THE INVENTION

The present invention now provides for the first time the geneticsequence of the major grass pollen allergen Phl p 4, with three dominantsequences (SEQ ID NOS: 1, 3 and 5) arising from the single nucleotidepolymorphisms (SNPs) found.

The present invention therefore relates to a DNA molecule correspondingto a nucleotide sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO: 3 and SEQ ID NO: 5 or a DNA molecule corresponding to anucleotide sequence which encodes for the major allergen Phl p 4 fromPhleum pratense.

The invention also covers fragments, new combinations of partialsequences and point mutants having a hypoallergenic action.

The invention therefore furthermore relates to corresponding partialsequences, a combination of partial sequences or exchange, eliminationor addition mutants which encode for an immunomodulatory,T-cell-reactive fragment of a group 4 allergen of the Poaceae.

In addition to the group 4 allergens of the other grass species, thegroup 13 allergens are also of interest in connection with the presentinvention since they exhibit a very similar molecular weight to thegroup 4 allergens in the SDS-PAGE and are difficult to separate bybiochemical techniques (Suck et al., 2000, Clin. Exp. Allergy 30:324-332, Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402). With theaid of the protein and DNA sequence according to the invention which isnow available for the first time, however, it can unambiguously be shownthat groups 4 and 13 have significantly different amino acid sequences.

With knowledge of the DNA sequence of naturally occurring allergens, itis now possible to prepare these allergens as recombinant proteins whichcan be used in the diagnosis and therapy of allergic diseases (Scheinerand Kraft, 1995, Allergy 50: 384-391).

A classical approach to effective therapeutic treatment of allergies isspecific immunotherapy or hypo-sensitization (Fiebig, 1995, Allergo J. 4(6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102(4):558-562). In this method, the patient is injected subcutaneously withnatural allergen extracts in increasing doses. However, there is a riskin this method of allergic reactions or even anaphylactic shock. Inorder to minimize these risks, innovative preparations in the form ofallergoids are being employed. These are chemically modified allergenextracts which have significantly reduced IgE reactivity, but identicalT-cell reactivity compared with the untreated extract (Fiebig, 1995,Allergo J. 4 (7): 377-382).

Even more substantial therapy optimization would be possible withallergens prepared by recombinant methods. Defined cocktails ofhigh-purity allergens prepared by recombinant methods, optionallymatched to the individual sensitization patterns of the patients, couldreplace extracts from natural allergen sources since these, in additionto the various allergens, contain a relatively large number ofimmunogenic, but non-allergenic secondary proteins.

Realistic perspectives which may result in reliable hypo-sensitizationwith expression products are offered by specifically mutated recombinantallergens in which IgE epitopes are specifically deleted withoutimpairing the T-cell epitopes which are essential for therapy (Schrammet al., 1999, J. Immunol. 162: 2406-2414).

A further possibility for therapeutic influencing of the disturbedTH-cell equilibrium in allergy sufferers is immunotherapeutic DNAvaccination. This involves treatment with expressible DNA which encodesfor the relevant allergens. Initial experimental evidence ofallergen-specific influencing of the immune response has been furnishedin rodents by injection of allergen-encoding DNA (Hsu et al., 1996,Nature Medicine 2 (5): 540-544).

The present invention therefore also relates to a DNA molecule describedabove or below or a corresponding recombinant expression vector asmedicament.

The corresponding proteins prepared by recombinant methods can beemployed for the therapy and for the in vitro and in vivo diagnosis ofpollen allergies.

For preparation of the recombinant allergen, the cloned nucleic acid isligated to an expression vector, and this construct is expressed in asuitable host organism. After biochemical purification, this recombinantallergen is available for the detection of IgE antibodies by establishedmethods.

The present invention therefore furthermore relates to a recombinantexpression vector comprising a DNA molecule described above or below,functionally linked to an expression control sequence and a hostorganism transformed with the said DNA molecule or the said expressionvector.

The invention likewise relates to the use of at least one DNA moleculedescribed above or at least one expression vector described above forthe preparation of a medicament for immunotherapeutic DNA vaccination ofpatients having allergies in the triggering of which group 4 allergensof the Poaceae are involved and/or for the prevention of such allergies.

As already stated, the invention can be used as an essential componentin a recombinant allergen- or nucleic acid-containing preparation forspecific immunotherapy. There are a number of possibilities here.Firstly, the protein with an unchanged primary structure may be aconstituent of the preparation. Secondly, through specific deletion ofIgE epitopes of the entire molecule or the preparation of individualfragments which encode for T-cell epitopes, a hypoallergenic(allergoidal) form can be used in accordance with the invention fortherapy in order to prevent undesired side effects. Finally, the nucleicacid per se, if ligated with a eukaryotic expression vector, gives apreparation which on direct application modifies the allergic immunestate in the therapeutic sense.

The invention thus relates to recombinant DNA molecules corresponding toSEQ ID NOS: 1, 3 or 5, where the nucleotide sequence of positions 1-69has been derived from the amino acid sequence of the Phl p 4 N-terminus.Codons which frequently occur in E. coli were used here. From position70, the DNA sequence corresponds to that which has been identified ingenomic and cDNA of Phleum pratense.

The present invention therefore furthermore relates to a DNA moleculecomprising a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5, commencing with position 70, which encodes for apolypeptide having the properties of the major allergen Phl p 4 fromPhleum pratense.

Furthermore, the present invention relates to the polypeptides encodedby one or more of the above-described DNA molecules, preferably in theirproperty as medicament.

These are, in particular, polypeptides according to SEQ ID NO: 2, SEQ IDNO: 4 or SEQ ID NO: 6, where amino acid positions 1-33 have beendetermined by N-terminal amino acid sequencing of the isolated naturalPhl p 4 allergen. Positions 24-500 were derived from the DNA sequenceaccording to SEQ ID NOS: 1, 3 and 5. Variable amino acids at positions6, 7, 8 and 9 originate from the N-terminal protein sequencing ofvarious preparations of natural Phl p 4 (Table 1).

Accordingly, the invention also relates to a process for the preparationof polypeptides of this type by cultivation of a host organism accordingto claim 11 and isolation of the corresponding polypeptide from theculture.

The invention likewise relates to the use of at least one polypeptidedescribed above for the preparation of a medicament for the diagnosisand/or treatment of allergies in the triggering of which group 4allergens of the Poaceae are involved and for the prevention of suchallergies.

These polypeptides or proteins according to the invention which act asallergens for humans are present in the pollen grains of Phleumpratense. The pollen grains of the other Poaceae species, such as, forexample, Lolium perenne, Dactylis glomerata, Poa pratensis, Cynodondactylon, Holcus lanatus, inter alia, contain homologous allergenmolecules (group 4 allergens).

The homology of these molecules has been demonstrated through theirimmunological cross-reactivity both with murine monoclonal antibodiesand also with human IgE antibodies.

Consequently, the invention also relates to sequences which arehomologous to the Phl p 4 DNA sequence and corresponding DNA moleculesof group 4 allergens from other Poaceae such as, for example, Loliumperenne, Dactylis glomerata, Poa pratensis, Cynodon dactylon, Holcuslanatus, Triticum aestivum and Hordeum vulgare, which, owing to thesequence homology which exists, hybridize with Phl p 4 DNA understringent conditions or have immunological cross-reactivity with respectto Phl p 4.

The following procedure was followed in the determination of the proteinand DNA sequence of Phl p 4:

The natural allergen Phl p 4 was purified and isolated by describedmethods (Fahlbusch et al. 1998, Clin. Exp. Allergy 28: 799-807, Suck etal. 2000, Clin. Exp. Allergy 30: 1395-1402). The micropurification andthe removal of traces of the group 13 allergen was carried out by themethod described by Suck et al. (2000, Clin. Exp. Allergy 30:1395-1402).

The N-terminal amino acid sequence of this Phl p 4 isolated from Phleumpretense was determined by means of Edman degradation. The N-terminalsequences (P1a-f shown in Table 1 were determined with various batchesof Phl p 4. The consensus sequence for the first 15 positions isregarded as being the following sequence: YFPP′P′AAKEDFLGXL (SEQ ID NO:33). Position 14 could not be determined; it is probably occupied bycysteine. The different amino acids in positions 6, 7, 8 and 9 in thedifferent batches indicate variations in the sense of isoforms.Positions 4 and 5 are occupied by hydroxyproline (P′), which wasunambiguously determined by specific analysis in the analyses ofpreparations p1-a and -b.

Treatment of the SDS-denatured Phl p 4 with the endopeptidase Glu-C(Promega, Heidelberg, Germany) gave various peptides. The amino acidsequences shown in Table 1 were determined for two peptides (P2 and P3).2 peptides (P4 and P5) were purified by cleavage using the endopeptidaseLys-C (Roche, Mannheim, Germany) and sequenced (Table 1). A furtherpeptide (P6) was isolated by CNBr cleavage and the amino acid sequencewas determined (Table 1).

The amino acid sequences of the N-terminal sequence and the internalpeptides 2 and 6 were used as the basis for the construction ofdegenerated primers. Amplicons were prepared with the sense primer No.30 and the antisense primer No. 37 (Table 2) using genomic DNA fromPhleum pratense. The clones obtained from these amplicons were sequenced(FIG. 1) and used for the construction of the specific sense primer No.82 (Table 2). Using a cDNA prepared from the representative mRNApopulation from Phleum pretense pollen and the specific sense primer No.82 according to the invention and the anchor primer AUAP (LifeTechnologies, Karlsruhe, Germany), a PCR was carried out under stringentconditions. This approximately 450 kb amplicon was sequenced and themissing sequence as far as the 3′ end of the Phl p 4 gene was thusidentified (FIG. 2). Based on this C-terminal Phl p 4 sequencedetermined in accordance with the invention, the specific antisenseprimers No. 85 and No. 86 were constructed (Table 2). Based on theN-terminal amino acid sequence of the Phl p 4 peptide P1-a (Table 1),the degenerated sense primer No. 29, derived from the DNA encoding foramino acid positions 24-33 (LYAKSSPAYP (SEQ ID NO: 34)), wasconstructed.

A PCR was carried out with primers No. 29 and No. 86 using genomicPhleum pratense DNA. This PCR product was employed as the basis for asecond PCR (nested PCR) with primers No. 29 and No. 85. The ampliconswere inserted into the vector pGEM T-easy (Promega, Heidelberg,Germany), cloned and sequenced. This sequence begins at position 24calculated from the N-terminus or position 70 of the DNA sequence inaccordance with SEQ ID NOS: 1, 3 or 5 and extends to primer No. 85(position 1402 in SEQ ID NOS: 1, 3 or 5), which is localized in thealready determined C-terminal section of the Phl p 4 gene. Using thesedata, the complete amino acid sequence of the Phl p 4 molecule can beconstructed from the first 33 amino acid positions, determined byprotein sequencing, and the deduced amino acid sequence (477 positions),which can be derived from the clones prepared with primers No. 29/No. 85and No. 82/anchor primer. The two clones overlap in 197 positions oftheir nucleotide sequence. The peptide encoded by clone No. 29/No. 85overlaps in 10 amino acid positions with the N-terminal sequence(positions 1-33), determined by direct amino acid sequencing, of Phl p4, where the amino acids determined by the two methods correspond.

The amino acid sequence of Phl p 4 based on the directly determinedN-terminal amino acids and the deduced amino acid sequence correspondsto the sequences listed in the sequence protocol under SEQ ID NOS: 2, 4and 6.

PCR products were prepared with the specific sense primer No. 88 (Table2) and the specific antisense primer No. 86 both using genomic and usingcDNA from Phleum pratense and sequenced directly.

This enables PCR errors to be excluded and genetic variations (singlenucleotide polymorphisms) to be discovered.

The single nucleotide polymorphisms found for the DNA sequence SEQ IDNO: 1 are shown in Table 3. Some of these single nucleotidepolymorphisms result in modified amino acids. These are shown in Table4. Furthermore, DNA clones which result in deviating amino acids withrespect to the dominant sequences SEQ ID NOS: 2, 4 and 6 were sequenced(Table 5). These amino acid variations are to be regarded as isoforms ofthe Phl p 4 molecule. The existence of such isoforms is to be expectedowing to the heterogeneous isoelectric behavior of natural Phl p 4. Allpollen allergens known hitherto have such isoforms. The fact that theDNA fragment determined with primers No. 29 and 86 actually encodes fora protein which is identical with the natural Phl p 4 allergen can alsobe demonstrated, inter alia, by the fact that homologous peptidesequences in the deduced amino acid sequence of the recombinant Phl p 4molecule according to the invention are found (FIG. 3) for theidentified internal peptides P3, P4 and P5 (Table 1) of natural Phl p 4.The Phl p 4 amino acid sequence described shows that it is a basicmolecule having a calculated isoelectric point of 8.99 (SEQ ID NO: 2),8.80 (SEQ ID NO: 4) or 9.17 (SEQ ID NO: 6), consisting of 500 aminoacids. The quantitative amino acid composition is shown in Table 6. Thecalculated molecular weight of recombinant Phl p 4 is 55.762 (SEQ ID NO:2), 55.734 (SEQ ID NO: 4) or 55.624 (SEQ ID NO: 6) daltons. Thiscalculated molecular weight agrees very well with the molecular weightof natural Phl p 4 of 55 kDa determined by SDS-PAGE (Fahlbusch et al.,1998, Clin. Exp. Allergy 28: 799-807 and Suck et al., 2000, Clin. Exp.Allergy 30: 1395-1402).

Molecular weights of between 50 and 60 kDa have also been described forthe group 4 allergens of related grass species (Su et al., 1991, Clin.Exp. Allergy 21: 449-455; Jaggi et al., 1989, Int. Arch. Allergy Appl.Immunol. 89: 342-348; Jaggi et al., 1989, J. Allergy Clin. Immunol. 83:845-852; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; 14-17).

For the preparation of the recombinant Phl p 4 protein, the DNA sequenceaccording to SEQ ID NOS: 1, 3 and/or 5 encoding for Phl p 4 was insertedinto expression vectors (for example PPROEX, pλCro, pSE 380). For theN-terminal amino acids known from protein sequencing, E. coli optimizedcodons were used.

After transformation into E. coli, expression and purification of therecombinant Phl p 4 by various separation methods, the resultant proteinwas subjected to a refolding process.

This rPhl p 4 protein obtained in this way gives a single band in theSDS-PAGE which covers the same molecular weight range as natural Phl p4. The immunological reactivity of rPhl p 4 has been demonstrated byreaction with the murine monoclonal antibodies 5H1 and 3C4, which hadbeen induced using natural Phl p 4 and cross-react with the homologousproteins (group 4) of the Poaceae (Fahlbusch et al., 1998, Clin. Exp.Allergy 28:799-807; Gavrovi-Jankulovi et al., 2000, Invest. Allergol.Clin. Immunol. 10 (6): 361-367) (FIG. 4). rPhl p 4 reacts with IgEantibodies of allergy sufferers which have demonstrated IgE reactivitywith natural Phl p 4. This IgE reactivity and thus the action asallergen have been demonstrated both in the dot test, Western blot andalso after adsorption of the allergen on polystyrene microtitre plates.Detection by Western blot is shown in FIG. 5. On reaction of rPhl p 4with basophiles of allergen group 4-reactive grass pollen allergysufferers, these are stimulated to increased expression of theactivation marker CD 203c. This basophile activation by rPhl p 4 clearlyshows that this molecule also acts functionally as an allergen.

This rPhl p 4 allergen can thus be employed for the highly specificdiagnosis of grass pollen allergy sufferers. This diagnosis can becarried out in vitro by detection of specific antibodies (IgE, IgG1-4,IgA) and reaction with IgE-loaded effector cells (for example basophilesfrom the blood) or in vivo by skin test reactions and provocation at thereaction organ.

The reaction of rPhl p 4 with T-lymphocytes of grass pollen allergysufferers has been detected by allergen-specific stimulation of theT-lymphocytes for proliferation and cytokine synthesis both with T-cellsin freshly prepared blood lymphocytes and on established nPhl p4-reactive T-cell lines and clones.

Based on the rPhl p 4 DNA sequence described, partial sequences encodingfor peptides having from 50 to 350 amino acids were cloned intoexpression vectors. These partial sequences cover sequentially thecomplete sequence of rPhl p 4, with overlaps of at least 12 amino acidsoccurring. The expressed peptides correspond to Phl p 4 fragments. ThesePhl p 4 fragments do not react individually or as a mixture with the IgEantibodies of allergy sufferers or only do so to a small extent, so thatthey can be classified as hypoallergenic. In contrast, the mixture ofthese fragments is capable, in the same way as complete recombinant ornatural Phl p 4, of stimulating T-lymphocytes of grass pollen allergysufferers having Phl p 4 reactivity.

FIG. 4 shows as an example the characterization of two such Phl p 4fragments corresponding to amino acids 1-200 and 185-500 by binding toPhl p 4-specific monoclonal mouse antibodies. The C-terminal fragment185-500 reacts only with monoclonal antibody 5H1, while the N-terminalfragment 1-200 clearly reacts with monoclonal antibody 3C4. It can beseen from FIG. 5 that fragment 185-500 reacts less strongly with the IgEfrom the sera of allergy sufferers B and C, i.e. is less allergenic thanfragment 1-200, which has reduced IgE reactivity (hypoallergeneity), atleast to patient serum C.

The present invention therefore also relates to a DNA molecule describedabove or below, encoding for a fragment 1-200, with amino acids 1-200 ofPhl p 4, and a DNA molecule encoding for a fragment 285-500, with aminoacids 285-500 of Phl p 4.

The triplets encoding for the cysteines were modified by site-specificmutagenesis in such a way that they encode for other amino acids,preferably serine. Both variants in which individual cysteines have beenreplaced and those in which various combinations of 2 cysteine radicalsor all 5 cysteines have been modified have been prepared. The expressedproteins of these cysteine point mutants have highly reduced or zeroreactivity with IgE antibodies of allergy sufferers, but react with theT-lymphocytes of these patients. The present invention thereforefurthermore relates to a DNA molecule described above or below in whichone, more or all of the cysteine radicals of the correspondingpolypeptide have been replaced by another amino acid by site-specificmutagenesis.

The immunomodulatory activity of the hypoallergenic fragments whichcorrespond to polypeptides having T-cell epitopes and those of thehypoallergenic point mutants (for example cysteine polymorphisms) hasbeen demonstrated by reaction thereof with T-cells of grass pollenallergy sufferers.

Such hypoallergenic fragments or point mutants of the cysteines can beemployed as preparations for the hypo sensitization of allergy suffererssince they react with equal effectiveness with the T-cells, but, owingto the reduced or entirely absent IgE reactivity, result in reducedIgE-mediated side effects.

If the nucleic acids encoding for the hypoallergenic Phl p 4 variants orthe unmodified DNA encoding for Phl p 4 are ligated with a humanexpression vector, these constructs can likewise be used as preparationsfor immuno-therapy (DNA vaccination).

Finally, the present invention relates to pharmaceutical compositionscomprising at least one DNA molecule described above or at least oneexpression vector described above and optionally further activeingredients and/or adjuvants for immunotherapeutic DNA vaccination ofpatients having allergies in the triggering of which group 4 allergensof the Poaceae are involved and/or for the prevention of such allergies.

A further group of pharmaceutical compositions according to theinvention comprises, instead of the DNA, at least one polypeptidedescribed above and is suitable for the diagnosis and/or treatment ofthe said allergies.

Pharmaceutical compositions in the sense of the present inventioncomprise, as active ingredients, a polypeptide according to theinvention or an expression vector and/or respective pharmaceuticallyusable derivatives thereof, including mixtures thereof in all ratios.The active ingredients according to the invention can be brought hereinto a suitable dosage form together with at least one solid, liquidand/or semi-liquid excipient or adjuvant and optionally in combinationwith one or more further active ingredients.

Particularly suitable adjuvants are immunostimulatory DNA oroligonucleotides having CpG motives.

These compositions can be used as therapeutic agents or diagnosticagents in human or veterinary medicine. Suitable excipients are organicor inorganic substances which are suitable for parenteral administrationand do not adversely affect the action of the active ingredientaccording to the invention. Particularly suitable for parenteraladministration are solutions, preferably oil-based or aqueous solutions,furthermore suspensions, emulsions or implants. The active ingredientaccording to the invention may also be lyophilized and the resultantlyophilisates used, for example, for the preparation of injectionpreparations. The compositions indicated may be sterilized and/orcomprise adjuvants, such as lubricants, preservatives, stabilizersand/or wetting agents, emulsifiers, salts for modifying the osmoticpressure, buffer substances and/or a plurality of further activeingredients.

Furthermore, sustained-release preparations can be obtained bycorresponding formulation of the active ingredient according to theinvention.

The invention thus also serves for improving in vitro diagnosis as partof allergen component-triggering identification of the patient-specificsensitization spectrum. The invention likewise serves for thepreparation of significantly improved preparations for the specificimmunotherapy of grass pollen allergies.

TABLE 1 Amino acid sequence of Phl p4 peptides SEQ Peptide IDAmino acids Preperation batch NO 1 6 11 16 21 26 31 Intact Phl p4 P1-a35 YPPP′P′ AAKED FLGXL VKEIP PRLLY AKSSP AVP P1-b 36 YFPP′P′ AAKED FLGXLVKE-P PRLLY AKSSP P1-c 37 YRPXX AAKED FLGXL P1-d 38 YPPXX AKEED FLGXLP1-e 39 YFPXX AAKDD FLGXL P1-f 40 YPPXX LAMED F Glu-C P2 41 SATPF KHREGVLFNI QTV fragments P3 42 GLXYR XLXPE Lys-C P4 43 KXMGD DNFXA VRfragments P5 44 APEGA VDI I  CNBr P6 45 MEPYV SINPV QAYAE Y fragment

TABLE 2 Degenerated and specific sense and antisenseprimers constructed on the basisof Phl p 4peptide sequences and DNA sequences Sense/ SEQ Primer Peptide/ anti- IDNo. DNA sense NO Nucleotide sequence 29 Phl p 4-P1 s 46YTN TAY GCN AAR WSN WSN CCN GCN TAY CC 30 Phl P 4-P2 s 47CAY WGN AAR GGN GTN YTN TTY AAY ATN C 37 Phl p 4-P6 as 48TAR TTN GCH TAN GCT TGN ACN GGR TT 82 Phl p 4- s 49 ACT ACT GGT TCG CCCDNA-NYW CGG GAG CC 85 Phl p 4- as 50 TGA AGT ATT TCT GGC DNA-GLVCCT ACA CCA AAC C 86 Phl p 4- as 51 CCC TTG GTG ATG GCG DNA-QRLAGC CTC TGG 88 Phl p 4- s 52 CTC AGT CCT GGG GCA DNA-PSV GAC CAT CC

The nucleotide sequences of primers 82, 85, 86 and 88 is shown in theusual 4-letter code. In the case of primers 29, 30 and 37, the IUPAC-IUBDNA code is used; the letter ‘N’ here stands for inosine.

TABLE 3 Detected single nucleotide polymorphisms Position in Nucleotideaccording to Detected sequence SEQ ID NO 1 SNPs 85 T A 130 C A 159 G A160 A C 169 G A 185 C T 186 C A 222 G C 226 G A 227 G C 228 T C 237 C T273 C T 285 C T 286 C T 298 G A 299 A C 303 C T 309 C G 318 T C 320 G A333 C G 348 G C 369 C G 409 C T 411 C T 420 T C 421 A C 423 A C 424 G A425 T C 456 C G 462 C A 522 G C 525 C G 567 G A 618 C T 655 A C 657 G A662 G A 680 C T 684 G C 690 C A 691 G A 693 G A 703 C T, A 710 A C 711 GA 713 C T 743 G A 750 G A 768 C T 773 A C 790 G A 798 G C 801 G A 804 CG 809 C A 834 G C 844 C A 859 A T 865 A G 879 G C 895 G C 900 G C, A 918G A 961 A G 962 A C 964 A C 987 G C 994 A T 1020 G A 1023 G C 1036 G C1040 C T 1041 G C 1047 C A 1051 A G 1052 G A, C 1053 G A, C, T 1056 G C1069 T C 1073 G A 1084 C G 1086 G C 1090 C T 1098 G C 1151 G C 1152 G C1155 G C 1161 G C 1185 C G 1229 G C 1233 G C 1239 A C 1240 T C 1242 G C1257 G C 1266 C T 1269 C T 1278 A C, G 1305 C G 1308 C T 1311 C A 1335 GC 1350 G C 1357 T A 1359 A G 1370 G C 1377 T C 1378 T A 1379 T A 1383 GC 1398 C T 1411 T C 1414 C G 1425 C A 1428 C T 1443 G C 1449 C T 1464 GA 1485 G A 1498 A C

TABLE 4 Amino acid exchanges as a consequence of single nucleotidepolymorphisms Position in Amino acid according to Detected sequence SEQID NO 2 exchanges 6 A L 7 A K 8 K N 9 E D 29 S T 54 I L 57 V I 62 A V 76G T, N, S 100 E T 107 S N 137 H Y 141 T P 142 V A, T 189 T K 219 K Q 221R K 227 P L 231 V I 235 P T, S 237 K T 238 A V 248 R K 258 D A 264 V I270 T K 282 Q K 287 M L 289 S G 299 A P 321 N A 322 I L 332 T S 346 E Q347 P L 351 R E, T 357 F L 358 S N 362 L V 364 P S 384 W S 410 G A 419 ED 456 F Y 457 S A, N 460 L K 468 K M 472 Q E 498 K Q

TABLE 5 Deviating amino acid positions in individual recombinant Phl p 4clones compared with SEQ ID NO 2 Example Deviating positions* Clone 1L54, I57, V62, S76, T100, N107, Y137, P141, T142, K189, Q219, K221,L227, I231, S235, T237, V238, K248, A258, I264, K270, K282, L287, P299,A321, L322, S332, Q346, P347, T351, L357, N358, V362, S384, A410, D419,Y456, A457, K460, E472 Clone 2 L54, I57, V62, T76, T100, N107, Y137,P141, T142, K189, Q219, K221, I231, S235, T237, V238, K248, A258, I264,K270, K282, L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358,V362, S384, A410, D419, Y456, A457, K460, E472 Clone 3 P141, K282, L287,P299, L347, E351 Clone 4 G289, A410, D419, Y456, A457, K460, E472 Clone5 L347, E351, S384, A410, D419, Y456, A457, K460, E472 Clone 6 N107,Y137, P141, T142, K189, Q219, K221, I231, S235, T237, V238, K248, A258,I264, K270, K281, L287, P299, A321, L322, S332, Q346, P347, T351, L357,N358, V362, S384, A410, D419, Y456, A457, K460 Clone 7 K248, A258, I264,K270, K282, L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358,V362, S384 Clone 8 Q219, K221, I231, S235, T237, V238, K248, A258, I264,K270, K282, L287, P299, E351 Clone 9 M231, T246, A251, C263, G289, L307,L309, E334 Clone 10 Q219, K221, I231, S235, T237, M238, V242, V246,K248, A258, I264, K270, K282, L287, P299, A321, L322, S332, Q346, P347,T351, N358, V362, S384, insertion of GA between positions 407 and 408,N452, Y456, A457, K460, E472 Clone 11 Insertion of GA between positions407 and 408 *[Amino acid according to SEQ ID NO 2/position insequence/deviating amino acid]

TABLE 6 Amino acid composition of Phl 4 Amino acids Number % by weightCharged 138/138/138 33.89/33.86/33.93 Acid 45/46/43 9.82/10.05/9.38Basic 54/53/55 13.67/13.39/13.78 Polar 120/119/174 24.88/24.71/25.89Hydrophobic 180/180/180 35.64/35.66/35.43 A Ala 40/40/41 5.10/5.10/5.24C Cys 5/5/5 0.92/0.93/0.93 D Asp 24/24/24 4.95/4.96/4.97 E Glu 21/22/194.86/5.10/4.41 F Phe 24/24/22 6.33/6.34/5.82 G Gly 42/42/404.30/4.30/4.10 H His 10/10/9 2.46/2.46/2.22 I Ile 29/29/305.88/5.89/6.10 K Lys 29/29/33 6.67/6.67/7.60 L Leu 33/33/356.70/6.70/7.12 M Met 11/11/10 2.59/2.59/2.36 N Asn 22/22/234.50/4.50/4.72 P Pro* 38/39/39 6.62/6.80/6.81 Q Glu 15/15/153.45/3.45/3.46 R Arg 25/24/22 7.00/6.73/6.18 S Ser 32/32/335.00/5.00/5.17 T Thr 22/21/22 3.99/3.81/4.00 V Val 41/41/407.29/7.29/7.13 W Trp 13/13/12 4.34/4.34/4.02 Y Tyr 24/24/267.02/7.03/7.63 *including hydroxyproline

The values are given for the three dominant sequences in the order SEQID NO2/SEQ ID NO: 4/SEQ ID NO: 6.

The invention claimed is:
 1. A method for the treatment of allergies inthe triggering of which group 4 allergens of the Poaceae are involved ina subject in need thereof, comprising administering to said subject aneffective amount of the polypeptide encoded by a DNA sequence selectedfrom the group consisting of (a) a DNA molecule encoding the polypeptidesequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; (b) SEQ ID NO 1,SEQ ID NO 3 and SEQ ID NO 5; and (c) a DNA molecule encoding apolypeptide variant consisting of the polypeptide sequence of SEQ ID NO:2 with the amino acid variations set forth in clones 1 to 11, wherein(1) clone 1 consists of L54, I57, V62, S76, T100, N107, Y137, P141,T142, K189, Q219, K221, L227, I231, S235, T237, V238, K248, A258, 1264,K270, K282, L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358,V362, S384, A410, D419, Y456, A457, K460, and E472; (2) clone 2 consistsof L54, I57, V62, T76, T100, N107, Y137, P141, T142, K189, Q219, K221,I231, S235, T237, V238, K248, A258, I264, K270, K282, L287, P299, A321,L322, S332, Q346, P347, T351, L357, N358, V362, S384, A410, D419, Y456,A457, K460, and E472; (3) clone 3 consists of P141, K282, L287, P299,L347, and E351; (4) clone 4 consists of G289, A410, D419, Y456, A457,K460, and E472; (5) clone 5 consists of L347, E351, S384, A410, D419,Y456, A457, K460, and E472; (6) clone 6 consists of N107, Y137, P141,T142, K189, Q219, K221, I231, S235, T237, V238, K248, A258, 1264, K270,K282, L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358, V362,S384, A410, D419, Y456, A457, and K460; (7) clone 7 consists of K248,A258, 1264, K270, K282, L287, P299, A321, L322, S332, Q346, P347, T351,L357, N358, V362, and S384; (8) clone 8 consists of Q219, K221, I231,S235, T237, V238, K248, A258, 1264, K270, K282, L287, P299, and E351;(9) clone 9 consists of M231, T246, A251, C263, G289, L307, L309, andE334; (10) clone 10 consists of Q219, K221, I231, S235, T237, M238,V242, V246, K248, A258, 1264, K270, K282, L287, P299, A321, L322, S332,Q346, P347, T351, N358, V362, S384, insertion of GA between positions407 and 408, N452, Y456, A457, K460, and E472; and (11) clone 11consists of insertion of GA between positions 407 and
 408. 2. A methodfor the treatment of allergies in the triggering of which group 4allergens of the Poaceae are involved in a subject in need thereof,comprising administering to said subject an effective amount of thepolypeptide encoded by a DNA sequence which encodes an isolatedpolypeptide which comprises (a) a single nucleotide polymorph of thepolynucleotide sequence of SEQ ID NO: 1, or (b) a single amino acidpolymorph of the polypeptide sequence of SEQ ID NO:
 2. 3. A method forthe treatment of allergies in the triggering of which group 4 allergensof the Poaceae are involved in a subject in need thereof, comprisingadministering to said subject an effective amount of animmunomodulatory, T cell-reactive Phl p 4 polypeptide fragment encodedby a DNA sequence selected from the group consisting of (a) a DNAmolecule encoding a polypeptide consisting of amino acids 1-200 of thepolypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or apolypeptide variant consisting of the polypeptide sequence of SEQ ID NO:2 with the amino acid variations of clones 1 to 11, wherein (1) clone 1consists of L54, I57, V62, S76, T100, N107, Y137, P141, T142, K189,Q219, K221, L227, I231, S235, T237, V238, K248, A258, 1264, K270, K282,L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358, V362, 5384,A410, D419, Y456, A457, K460, and E472; (2) clone 2 consists of L54,I57, V62, T76, T100, N107, Y137, P141, T142, K189, Q219, K221, I231,S235, T237, V238, K248, A258, 1264, K270, K282, L287, P299, A321, L322,S332, Q346, P347, T351, L357, N358, V362, S384, A410, D419, Y456, A457,K460, and E472; (3) clone 3 consists of P141, K282, L287, P299, L347,and E351; (4) clone 4 consists of G289, A410, D419, Y456, A457, K460,and E472; (5) clone 5 consists of L347, E351, S384, A410, D419, Y456,A457, K460, and E472; (6) clone 6 consists of N107, Y137, P141, T142,K189, Q219, K221, I231, S235, T237, V238, K248, A258, I264, K270, K282,L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358, V362, S384,A410, D419, Y456, A457, and K460; (7) clone 7 consists of K248, A258,I264, K270, K282, L287, P299, A321, L322, S332, Q346, P347, T351, L357,N358, V362, and S384; (8) clone 8 consists of Q219, K221, I231, S235,T237, V238, K248, A258, I264, K270, K282, L287, P299, and E351; (9)clone 9 consists of M231, T246, A251, C263, G289, L307, L309, and E334;(10) clone 10 consists of Q219, K221, I231, S235, T237, M238, V242,V246, K248, A258, I264, K270, K282, L287, P299, A321, L322, S332, Q346,P347, T351, N358, V362, S384, insertion of GA between positions 407 and408, N452, Y456, A457, K460, and E472; and (11) clone 11 consists ofinsertion of GA between positions 407 and 408; and (b) a DNA moleculeencoding a polypeptide consisting of amino acids 185-500 of thepolypeptide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or apolypeptide variant consisting of the polypeptide sequence of SEQ ID NO:2 with the amino acid variations of clones 1 to 11, wherein (1) clone 1consists of L54, I57, V62, S76, T100, N107, Y137, P141, T142, K189,Q219, K221, L227, I231, S235, T237, V238, K248, A258, I264, K270, K282,L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358, V362, 5384,A410, D419, Y456, A457, K460, and E472; (2) clone 2 consists of L54,I57, V62, T76, T100, N107, Y137, P141, T142, K189, Q219, K221, I231,S235, T237, V238, K248, A258, I264, K270, K282, L287, P299, A321, L322,S332, Q346, P347, T351, L357, N358, V362, S384, A410, D419, Y456, A457,K460, and E472; (3) clone 3 consists of P141, K282, L287, P299, L347,and E351; (4) clone 4 consists of G289, A410, D419, Y456, A457, K460,and E472; (5) clone 5 consists of L347, E351, S384, A410, D419, Y456,A457, K460, and E472; (6) clone 6 consists of N107, Y137, P141, T142,K189, Q219, K221, I231, S235, T237, V238, K248, A258, I264, K270, K282,L287, P299, A321, L322, S332, Q346, P347, T351, L357, N358, V362, S384,A410, D419, Y456, A457, and K460; (7) clone 7 consists of K248, A258,I264, K270, K282, L287, P299, A321, L322, S332, Q346, P347, T351, L357,N358, V362, and S384; (8) clone 8 consists of Q219, K221, I231, S235,T237, V238, K248, A258, I264, K270, K282, L287, P299, and E351; (9)clone 9 consists of M231, T246, A251, C263, G289, L307, L309, and E334;(10) clone 10 consists of Q219, K221, I231, S235, T237, M238, V242,V246, K248, A258, I264, K270, K282, L287, P299, A321, L322, S332, Q346,P347, T351, N358, V362, S384, insertion of GA between positions 407 and408, N452, Y456, A457, K460, and E472; and (11) clone 11 consists ofinsertion of GA between positions 407 and 408.